In the proposed project, we will continue to design, construct and characterize genetic circuits. We will use microfluidic tools to grow and observe single cells in precisely controlled environmental conditions. Single cell data will inform a set o mathematical models that will be used to identify key design characteristics, which will then be rigorously, tested using previously established molecular biology techniques. This multi- disciplinary approach will increase our understanding of gene regulation and lead to new tools for the synthetic biology community. Our first aim will be to explore the interaction of nested clocks. We previously constructed a robust intracellular clock and an intercellularly synchronized colony of clocks. Characterization of these systems revealed that the native enzymatic machinery induces a coupling between destabilized proteins that are \waiting to be degraded. In Aim 1, we will explore how such intracellular coupling can lead to clocks that are synchronized at multiple (intra- and intercellular) scales. In the next aim, we will explore the us of two intercellular coupling mechanisms to develop a new platform for synthetic biology. We have previously shown how quorum sensing and redox communication can be used to design a macroscopic (1cm) biosensor. In Aim 2, we will show how these coupling mechanisms can lead to an extremely stable toggle switch with switching transitions that are highly uniform at the single cell level. In the next aim, we will engineer light-sensitive circuits that produce complex spatiotemporal dynamics. Optogenetic circuits have recently been developed by several other groups and we plan to couple light-sensitive elements to our circuits to explore the light-guided propagation of signals throughout a spatially extended population of cells. In Aim 4, we will continue our work on a mammalian oscillator. Here, we will engineer a novel synthetic mammalian circuit that relies on a negative feedback mechanism that is mediated by a transrepressor that acts by inducing local chromatin remodeling upon binding the hybrid promoter. We will integrate our synthetic circuits into the cell genome in order to study how the molecular dynamics function within the chromosomal regulatory context. Finally, in Aim 5 we will develop bacterial minicells as a platform for delivering synthetic circuits to mammalian cells. To improve the functionality of minicells, we will construct and transfer to minicells an additional synthetic network that provides supplemental RNA polymerase, enabling independent gene expression long after minicell separation from parental bacteria. We will tailor our microuidic devices for housing and tracking minicells and will characterize circuit behavior using time-lapse uorescence microscopy. The successful completion of this project will lead to advances in our understanding of gene regulation and could ultimately result in the utilization of programmable logic in a gene-delivery context. 1